The present invention relates to the use of an adsorbent gel combining the properties of size exclusion and affinity chromatographies (AdSEC, for xe2x80x9cAdsorptive Size Exclusion Chromatographyxe2x80x9d).
The principle of an AdSEC gel results from the fusion of two chromatographic techniques: size exclusion and affinity, so as to obtain supports combining the most advantageous properties thereof.
Size exclusion chromatography (gel filtration) allows the separation of molecules according to their steric bulk alone during their passive diffusion in a molecular sieve (gel). The largest molecules cannot penetrate the crosslinked matrix and are consequently excluded more rapidly from the column. This technique possesses the characteristic feature of not exhibiting interactions between the support and the molecules, and therefore of being relatively only slightly sensitive to the biochemical conditions (pH, ionic strength) of the solution. On the other hand, because of its principle of diffusion, the limiting factors for its use are generally a long operation time (because low flow rates are used), as well as a relatively limited deposition of samples (1 to 5% of the column volume).
Affinity chromatography is based on molecular interactions between the support (matrix onto which affinity ligands are grafted) and the molecules to be separated. Among these affinity ligands, immobilized metal ions, introduced in 1975 by Porath et al. (Nature, 1975, 258, 598-599), represent a method of separation based on the interactions (coordination bonds) between biomolecules in solution and metal ions immobilized on a support; Zn(II), Cu(II), Ni(II) and Co(II) ions are the most commonly used. This is described as immobilized metal ion affinity chromatography (IMAC).
The combined use of the principles of size exclusion and affinity chromatographies (AdSEC) has been discussed by Porath et al. (Int. J. of Bio-Chromatogr., 1997, 3, 9-17). These authors have shown that iminodiacetic derivatives of dextran bearing metal ions as affinity ligand allow size exclusion and are capable of effectively concentrating solutions by their properties of adsorption and affinity. These authors have shown that an AdSEC gel column having a volume of 5 ml could bind a high percentage of compounds having a molecular weight of between 5 kDa and 50 kDa and concentrate them about 1000 fold in a single operation.
Such supports make it possible to adsorb the smallest molecules (having affinity for the grafted ligand) at high rates and volumes (not permitted in gel filtration). Moreover, during the synthesis of the adsorbent gel, the threshold of accessibility to the affinity ligand may be modulated during the synthesis of the gel according to the size of the biomolecule to be removed or to be purified.
Terminal renal insufficiency currently affects 22,000 people in France of which 20,000 are treated by iterative hemodialysis. Only 1800 can hope to undergo transplants each year, knowing that a quarter of them will return within 5 years to hemodialysis because of a rejection while waiting for a new transplant.
The survival of the uremic individual, all methods considered, can exceed 25 years if they do not suffer from a severe cardiovascular condition. In this case, the quality of survival is profoundly impaired over the years by the osteoarticular complications of terminal uremia, at the forefront of which there are described erosive arthropathies subsequent to depositions of xcex22-microglobulin (xcex22-M).
The mechanism of onset of these arthropathies begins as soon as the renal insufficiency responsible for accumulation of xcex22-microglobulin appears. This protein, having a molecular weight of 11,800 Da, will accumulate in the body over the years and become selectively deposited at the level of the cervical disks, of the shoulders, of the hips and of the wrists. Cardiac and digestive depositions have been reported. These depositions will make fragile the joint and the adjacent bone up to total destruction of the joint. Thus, a breakdown of the vertebral bodies is observed which can cause medullary compression with loss of control of the four members, irreversible articular luxations, loss of prehension in the hands and pseudofractures of the hip. Ductal nerve compressions are observed such as the carpal tunnel syndrome.
These complications irremediably lead the uremic individual toward invalidity and the bedridden state which conventional methods of dialysis cannot prevent. A transplant allows these lesions to be stabilized.
To effectively prevent these complications, it is important to be able to effectively purify the polluting components of blood, in particular xcex22-microglobulin, which are synthesized daily by the body and which are not, or not sufficiently, removed by the defective kidneys in dialyzed patients.
The purification of these various biomolecules can only be done on artificial membranes during dialysis, which are currently not sufficiently effective in spite of purification by filtration and nonspecific membrane adsorption.
The existing techniques for removing biomolecules, including xcex22-microglobulin, are currently of 3 types:
1. Removal of Biomolecules by Hemodialysis
Hemodialysis is a technique intended for subjects suffering from partial or complete renal insufficiency (FIG. 1). It consists in extracorporeal treatment of blood, providing the same functions as the kidney using a membrane process. The essential part of the hemodialyzer (1) is an exchange membrane, on either side of which circulate countercurrentwise the patient""s blood and the dialyzate obtained from the hemodialysis generator (2). This technique allows the purification of the small molecular weight compounds polluting the blood, such as urea, amino acids, inorganic salts, which are normally removed by the kidney. In the case of serum xcex22-microglobulin, the various dialysis membranes commonly used possess two antagonistic properties:
capture of xcex22-microglobulin by nonspecific adsorption on the membrane,
generation of xcex22-microglobulin by detachment of this molecule which is noncovalently associated with the surface of nucleated blood cells in the major histocompatibility complex type I.
The degree of generation of xcex22-microglobulin is one of the criteria which define the biocompatibility of the membranes. Thus, endowed with these two antagonist properties, some membranes lead overall, during a hemodialysis session, to an increase in the concentration of xcex22-microglobulin, whereas others reduce it.
However, regardless of the membranes used, these results level out over periods of over one year. Thus, it has been observed that the plasma level of xcex22-microglobulin in uremic patients after fifteen months of dialysis was invariably increased to be between 40 and 50 mg/l (against 1 to 2 mg/l in healthy patients). Such problems of biocompatibility also exist for the other biomolecules.
2. Removal of the Biomolocules by Hemofiltration
Once per month, the dialyzed individual is subjected to an ultrafiltration session. The module used (1) possesses a higher cut-off than in hemodialysis (average cut-off of 40 kDa) and allows the removal, by filtration, of the small molecules from plasma, including the smallest proteins, such as xcex22-microglobulin (FIG. 2). During an ultrafiltration session, the loss of plasma water is compensated by an equivalent supply of physiological saline (3).
The qualitative results, with respect to the removal of xcex22-microglobulin (purification and generation of this molecule by ultrafiltration membranes), are similar to those obtained in hemodialysis. There is thus a great influence of the nature of the membrane and of the duration of the hemofiltration. While some membranes appear to remove more xcex22-microglobulin over 5 hours (one session), a leveling out of the results is also observed over time. At the quantitative level, it appears that about 50% of the serum xcex22-microglobulin is removed per hemofiltration session. However, even if this technique is more effective for the purification of xcex22-microglobulin than hemodialysis, it remains inadequate for preventing and stopping the appearance of the disease. Furthermore, this technique has the disadvantage of removing numerous other small proteins apart from xcex22-microglobulin, since the ultrafiltrate is removed permanently.
3. Column/hemodialyzer Coupling
This method has been presented as an alternative to the customary hemodialysis and ultrafiltration methods (Nakazawa et al., Int. J. Artif. Organs, 1994, 17, 203-208). It consists in a serial adsorption of the biomolecules on a porous cellulose gel (350 ml of adsorbent), followed by conventional hemodialysis. In the case of xcex22-microglobulin, the gel is described as having a theoretical capacity for xcex22-microglobulin of 1 mg per ml of adsorbent. The results obtained are the best described in the literature, since in a patient in whom the initial xcex22-microglobulin level was 30 mg/l, this system made it possible to reduce the xcex22-microglobulin concentration to 10 mg/l final after 6 months of treatment. The authors presented an improvement in delaying the appearance of amyloid deposits in 2 cases out of 3, in their patients after therapy.
However, a drop in the concentration of some serum molecules (retinol binding protein, lysozymes) is also observed after treatment. This phenomenon is attributable to the direct passage of the blood through the adsorbent, which is likely to cause problems of biocompatibility.
Thus, the existing techniques for removing xcex22-microglobulin and other biomolecules have mainly two limits:
the biocompatibility of the supports, in particular for the generation of xcex22-microglobulin, that is to say the equilibrium between nonspecific adsorption on the membrane and the generation of xcex22-microglobulin during the passage of the cells in contact with them; this equilibrium determines the quantity of xcex22-microglobulin really removed during a hemodialysis or hemofiltration session.
the specificity of the substrate: indeed, the techniques of hemofiltration and of a specific binding with ligands coupled to gels lead to the undesirable removal of other molecules from serum.
A device for removing xcex22-microglobulin or any other biomolecule should therefore combine satisfactory (quantitative) removal with specific (qualitative) removal of the molecule in question.
In the present invention, the inventors therefore set themselves as objective:
the use, in a device intended to remove biomolecules, of an adsorbent gel combining the properties of size exclusion and affinity chromatographies, said gel essentially consisting of a polysaccharide matrix onto which is grafted a polymer coupled to an affinity ligand (AdSEC, for xe2x80x9cAdsorptive Size Exclusion Chromatographyxe2x80x9d gel) and having an adjustable cut-off of between 2 kDa and 60 kDa,
the use of an AdSEC gel for separating and purifying biomolecules having a molecular weight of between 2 kDa and 60 kDa,
a device intended for the removal of biomolecules having a molecular weight of between 2 kDa and 60 kDa comprising an ultrafiltration module optionally upstream and in series with a dialysis module and using an AdSEC gel column having an adjustable cut-off of between 2 kDa and 60 kDa, said column being mounted branching off from said ultrafiltration module; this device makes it possible to dispense with the problems of biocompatibility and to specifically remove the desired biomolecules,
a device for purifying biomolecules having a weight of between 2 kDa and 60 kDa using an AdSEC gel column having an adjustable cut-off of between 2 kDa and 60 kDa, said column optionally branching off from a filtration system; this device makes it possible to separate normal biomolecules and biomolecules modified for example by glycation.
In one advantageous embodiment, the polysaccharide matrix is agarose or is based on an agarose derivative, the polymer may be polyethylene glycol (PEG) or polypropylene glycol (PPG) and the affinity ligand may be, for example, a metal-chelating agent coupled to metal ions, a protein, a peptide, an enzyme substrate or an enzyme inhibitor.
In a preferred embodiment, the adsorbent gel consists of a matrix based on an agarose derivative onto which is grafted polyethylene glycol coupled to iminodiacetic acid (IDA) itself coupled to metal ions, for example copper(I) ions; this complex is called IMAdSEC (xe2x80x9cImmobilized Metal ion Adsorptive Size Exclusion Chromatographyxe2x80x9d) gel.
In an also preferred embodiment, the cut-off of the adsorbent gel is 20 kDa, thus allowing the removal or the purification of biomolecules whose molecular weight is less than 20 kDa, in particular serum xcex22-microglobulin.
The purification system according to the present invention possesses the characteristic feature of placing the adsorbent gel for the biomolecule to be removed branching from the circulation system for purifying. Thus, when blood is purified, there is at no time contact between the gel and the formed elements of the blood, therefore the problems of biocompatibility (for example generation of xcex22-microglobulin through contact with the nucleated cells of the blood) or hemolysis of the cells in contact with the gel are avoided.
Furthermore, unlike the other techniques currently used, the purification of the biomolecule to be removed or to be purified is carried out using a ligand which will retain only this molecule. This specificity is obtained by virtue of the double sieving of the ultrafiltration membrane (which retains for example the formed elements of the blood and the large serum molecules) and of the AdSEC gel which prevents access to the ligand for other molecules with affinity for the affinity ligand but whose size is greater than the cut-off of the gel.
The other advantage of the use of this AdSEC gel is its ease of regeneration. For example, when a metal is used as affinity ligand, it may be chelated by a solution of EDTA, which makes it possible to detach any molecule adsorbed onto the gel, thus allowing cleaning of the gel, its regeneration with a new metal load and its sterilization.
The removal system according to the invention may be used for example in the context of kidney dialysis; in this case, there is an additional advantage linked to the fact that the fraction purified by passage over the AdSEC gel returns to the patient, thus limiting losses of other elements present in the blood.